Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12616000417482
Ethics application status
Approved
Date submitted
29/03/2016
Date registered
1/04/2016
Date last updated
1/04/2016
Type of registration
Retrospectively registered

Titles & IDs
Public title
Exploration of alkaline supplements in healthy adults
Scientific title
Exploration of net endogenous non-carbonic acid production measurement, modulation and estimation using the Australian food database following administration of alkaline supplements in healthy adults
Secondary ID [1] 288858 0
nil
Universal Trial Number (UTN)
U1111-1181-3527
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
The problem studied was the measurement, modulation and estimation of net endogenous non-carbonic acid production in healthy people 298154 0
Condition category
Condition code
Diet and Nutrition 298318 298318 0 0
Other diet and nutrition disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
The study was a double blind, randomised, placebo controlled, cross-over trial with a four day wash out period between two phases. The wash out period was selected as large alkaline loads are known to be excreted within 1 – 2 days following their caseation.

The intervention consisted of a powdered fruit and vegetable supplement. 15g of the supplement was consumed in water 3 times daily for 3 days. To ensure compliance, the participants were met daily and questioned on their intake. The supplement was approved as safe by the University of the Sunshine Coast's Human Ethics committee (reference number: S/14/70). The supplement is also commercially available.

The following is the ingredients list printed on the nutritional supplement:
Certified Organic barley grass juice (5%)*, Certified Organic wheat grass juice (5%)*, Carrot, Apple fibre pectin, Lime, Pineapple, Apple, Kiwifruit, Prebiotic fibre (Fructooligosaccharides), Lemon, Calcium citrate, L-Glutamine, Natural fruit flavours, Green capsicum, Prebiotic fibre (Inulin), Broccoli sprouts, Certified Organic broken cell chlorella (2.5%)*, Magnesium citrate, Xylitol (natural sweetener derived from corn), Broccoli, Potassium bicarbonate, Guar gum, Brown seaweed extract (Fucoidan), Spinach, Bromelain, Daikon radish sprouts, Celery, Alfalfa sprouts, Parsley, Probiotic culture blend [1 million cfu/serve (Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum)], Dandelion leaves extract, Withania extract, Certified Organic spirulina (0.5%)*, Asparagus, Cucumber, Kelp extract, Aloe vera extract, Siberian ginseng extract, Shitake mushroom extract, Maitake mushroom extract, Natural flavour masker, Stevia extract, Chlorophyll, Black pepper extract.
*indicates certified organic by the Australian Certified Organic Standard
Intervention code [1] 294325 0
Treatment: Other
Comparator / control treatment
The placebo consisted of a look alike vegetable supplement. 15g of the supplement was consumed in water 3 times daily for 3 days. The supplement was approved as safe by the University of the Sunshine Coast's Human Ethics committee (reference number: S/14/70).

The following is the ingredients of the placebo:
Tapioca starch, asparagus powder, xylitol, natural lemon and lime flavour
Control group
Placebo

Outcomes
Primary outcome [1] 297792 0
Change in 24-hour urinary net acid excretion (NAE) as assessed by 3-step back titration. The methods are described below.

24-hr urine sample were analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius. Creatinine was measured spectrophotometrically by a colorimetric detection kit Cat no. 09151410 (Enzo Life Sciences, Farmingdale, USA) using a multiscan GO plate reader (Thermofisher Scientific, Waltham, USA). Ammonium was measured using methods of Rice et al by flow injection analysis using a Lachat QuikChem Registered Trademark 8000 (Lachat Instruments, Wisconsin, USA). Titratable acids and bicarbonate were measured in duplicate by methods of Litkowski and Wilson with the following modifications. Briefly, after addition of 0.05 M hydrogen chloride to 20 ml urine samples, simmering occurred for 20 minutes at 90 degrees Celsius. Concurrently, a blank was run in the same manner using MilliQ water (Milliport Corp., Bedford, USA). After samples cooled, bicarbonate was determined as the amount of 0.05 M sodium hydroxide necessary to titrate back to the initial pH at ambient temperature less the corresponding value for the blank. Thereafter, titratable acid was determined as the amount of 0.05 M sodium hydroxide necessary to titrate to pH 7.4 from the bicarbonate end point. The mean of the duplicates was used for the calculation of NAE and the intra-assay CV was 8.0 percent.
Timepoint [1] 297792 0
Difference between 24-hour net acid excretion on day 3 of the placebo as compared to day 3 of supplement.
Primary outcome [2] 297797 0
24-hour urine pH was analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius.
Timepoint [2] 297797 0
Difference between 24-hour pH on day 3 of the placebo as compared to day 3 of supplement.
Secondary outcome [1] 322272 0
Spot-tested urine pH in first morning void assessed using electronic Hanna HI98103 Checker Registered Trademark meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily.
Timepoint [1] 322272 0
Difference between first morning spot-test was taken on the morning of day 2, 3 and 4 during the placebo and supplement period.
Secondary outcome [2] 322277 0
Spot-tested urine pH in the pre-bed void assessed using electronic Hanna HI98103 Checker Registered Trademark meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily.
Timepoint [2] 322277 0
Difference between pre-bed spot-test was taken on the evening of day 1, 2 and 3 during the placebo and supplement period.
Secondary outcome [3] 322278 0
Spot-tested urine pH in second morning void assessed using electronic Hanna HI98103 Checker Registered Trademakr meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily.
Timepoint [3] 322278 0
Difference between the second morning spot-test was taken on the morning of day 4 during the placebo and supplement period.
Secondary outcome [4] 322294 0
In addition to the intervention study, this study also consisted of a validation study. That is, the accuracy and precision of net endogenous non-carbonic acid production as estimated by the Frassetto as well as the Remer and Manz equation was compared to the gold standard of 24-hour urinary net acid excretion. For the purposes for this form, these could be considered composite outcomes. To estimate the equation, weighted food diaries were collected and diet compositions were determined using Foodworks Professional (Xyris, Brisbane, Australia) using the Australian food database (NUTTAB 2010 Australian Government Nutrient Database, Canberra, Australia). When food items were missing, nutrients were entered according to nutrient information on the food label.
Timepoint [4] 322294 0
Equations were computed using dietary data from the concurrent 24-hr urinary collection periods which occurred on the third day of each phase.
Secondary outcome [5] 322298 0
In addition to the aforementioned validation study, this study also consisted of another validation component. That is, it sought to elucidate the relationship between spot-tested urine pH in the first morning void and 24-hour net acid excretion.

Spot-tested urine pH in first morning void was assessed using electronic Hanna HI98103 Checker Registered Trademakr meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily. To assess net acid excretion, 24-hr urine sample were analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius. Creatinine was measured spectrophotometrically by a colorimetric detection kit Cat no. 09151410 (Enzo Life Sciences, Farmingdale, USA) using a multiscan GO plate reader (Thermofisher Scientific, Waltham, USA). Ammonium was measured using methods of Rice et al by flow injection analysis using a Lachat QuikChem Registered Trademark 8000 (Lachat Instruments, Wisconsin, USA). Titratable acids and bicarbonate were measured in duplicate by methods of Litkowski and Wilson with the following modifications. Briefly, after addition of 0.05 M hydrogen chloride to 20 ml urine samples, simmering occurred for 20 minutes at 90 degrees Celsius. Concurrently, a blank was run in the same manner using MilliQ water (Milliport Corp., Bedford, USA). After samples cooled, bicarbonate was determined as the amount of 0.05 M sodium hydroxide necessary to titrate back to the initial pH at ambient temperature less the corresponding value for the blank. Thereafter, titratable acid was determined as the amount of 0.05 M sodium hydroxide necessary to titrate to pH 7.4 from the bicarbonate end point. The mean of the duplicates was used for the calculation of NAE and the intra-assay CV was 8.0 percent.
Timepoint [5] 322298 0
The spot-test occurred on the morning after the 24-hour urine collection which occurred on the third day of each phase.
Secondary outcome [6] 322300 0
In addition to the aforementioned validation component, this study also consisted of another validation component. That is, it sought to elucidate the relationship between spot-tested urine pH in second morning void and 24-hour net acid excretion.

Spot-tested urine pH in first morning void was assessed using electronic Hanna HI98103 Checker Registered Trademakr meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily. To assess net acid excretion, 24-hr urine sample were analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius. Creatinine was measured spectrophotometrically by a colorimetric detection kit Cat no. 09151410 (Enzo Life Sciences, Farmingdale, USA) using a multiscan GO plate reader (Thermofisher Scientific, Waltham, USA). Ammonium was measured using methods of Rice et al by flow injection analysis using a Lachat QuikChem Registered Trademark 8000 (Lachat Instruments, Wisconsin, USA). Titratable acids and bicarbonate were measured in duplicate by methods of Litkowski and Wilson with the following modifications. Briefly, after addition of 0.05 M hydrogen chloride to 20 ml urine samples, simmering occurred for 20 minutes at 90 degrees Celsius. Concurrently, a blank was run in the same manner using MilliQ water (Milliport Corp., Bedford, USA). After samples cooled, bicarbonate was determined as the amount of 0.05 M sodium hydroxide necessary to titrate back to the initial pH at ambient temperature less the corresponding value for the blank. Thereafter, titratable acid was determined as the amount of 0.05 M sodium hydroxide necessary to titrate to pH 7.4 from the bicarbonate end point. The mean of the duplicates was used for the calculation of NAE and the intra-assay CV was 8.0 percent.
Timepoint [6] 322300 0
The spot-test occurred on the morning after the 24-hour urine collection which occurred on the third day of each phase.
Secondary outcome [7] 322301 0
In addition to the aforementioned validation component, this study also consisted of another validation component. That is, it sought to elucidate the relationship between spot-tested urine pH in the last evening void and 24-hour net acid excretion.

Spot-tested urine pH in first morning void was assessed using electronic Hanna HI98103 Checker Registered Trademakr meters (Hanna instruments, Woonsocket, USA) which quantify pH within +/-0.1; these were calibrated daily. To assess net acid excretion, 24-hr urine sample were analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius. Creatinine was measured spectrophotometrically by a colorimetric detection kit Cat no. 09151410 (Enzo Life Sciences, Farmingdale, USA) using a multiscan GO plate reader (Thermofisher Scientific, Waltham, USA). Ammonium was measured using methods of Rice et al by flow injection analysis using a Lachat QuikChem Registered Trademark 8000 (Lachat Instruments, Wisconsin, USA). Titratable acids and bicarbonate were measured in duplicate by methods of Litkowski and Wilson with the following modifications. Briefly, after addition of 0.05 M hydrogen chloride to 20 ml urine samples, simmering occurred for 20 minutes at 90 degrees Celsius. Concurrently, a blank was run in the same manner using MilliQ water (Milliport Corp., Bedford, USA). After samples cooled, bicarbonate was determined as the amount of 0.05 M sodium hydroxide necessary to titrate back to the initial pH at ambient temperature less the corresponding value for the blank. Thereafter, titratable acid was determined as the amount of 0.05 M sodium hydroxide necessary to titrate to pH 7.4 from the bicarbonate end point. The mean of the duplicates was used for the calculation of NAE and the intra-assay CV was 8.0 percent.
Timepoint [7] 322301 0
The spot-test occurred on the evening of the 24-hour urine collection which occurred on the third day of each phase.
Secondary outcome [8] 322302 0
In addition to the aforementioned validation component, this study also consisted of another validation component. That is, it sought to elucidate the relationship between 24-hour urine pH at 36.5+/-0.5 degrees Celsius and 24-hour net acid excretion.

24-hr urine sample were analyzed by a calibrated labChem pH meter (TPS Pty Ltd, Melbourne, Australia) at ambient temperature and 36.5+/-0.5 degrees Celsius. Creatinine was measured spectrophotometrically by a colorimetric detection kit Cat no. 09151410 (Enzo Life Sciences, Farmingdale, USA) using a multiscan GO plate reader (Thermofisher Scientific, Waltham, USA). Ammonium was measured using methods of Rice et al by flow injection analysis using a Lachat QuikChem Registered Trademark 8000 (Lachat Instruments, Wisconsin, USA). Titratable acids and bicarbonate were measured in duplicate by methods of Litkowski and Wilson with the following modifications. Briefly, after addition of 0.05 M hydrogen chloride to 20 ml urine samples, simmering occurred for 20 minutes at 90 degrees Celsius. Concurrently, a blank was run in the same manner using MilliQ water (Milliport Corp., Bedford, USA). After samples cooled, bicarbonate was determined as the amount of 0.05 M sodium hydroxide necessary to titrate back to the initial pH at ambient temperature less the corresponding value for the blank. Thereafter, titratable acid was determined as the amount of 0.05 M sodium hydroxide necessary to titrate to pH 7.4 from the bicarbonate end point. The mean of the duplicates was used for the calculation of NAE and the intra-assay CV was 8.0 percent.
Timepoint [8] 322302 0
Data from the third day of each phase was used.

Eligibility
Key inclusion criteria
Participants were included if they were between 18–65 years old
Minimum age
18 Years
Maximum age
65 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
Participants were excluded if they used medication (except birth control), alkaline water or mineral and herbal supplements. Furthermore, those with a diagnosed health issue, BMI of <18 kg/m2, or pregnant were excluded.

Study design
Purpose of the study
Treatment
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Supplements were pre-weighed (15 g/ serve), sealed in silver bags and labelled A and B by a third party (Morlife). This concealed allocation to the investigator who generated the allocation sequence and enrolled the participants.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Participants were allocated by simple randomisation (computer generated list) to receive a powdered fruit and vegetable supplement or placebo.
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?
The people receiving the treatment/s
The people administering the treatment/s

Intervention assignment
Crossover
Other design features
The study was a double blind, randomised, placebo controlled, cross-over trial with a four day wash out period between two phases
Phase
Phase 1 / Phase 2
Type of endpoint/s
Efficacy
Statistical methods / analysis
Calculation of an a priori power using G-power 3 (Dusseldorf University, Dusseldorf, Germany) identified a required sample size of 11 participants to observe an expected change of 0.45 pH units in the first morning fasting urine following a potential renal acid load (PRAL) modulation of -23 mEq/d. Consequently, 16 adults were recruited to allow for attrition.

To determine whether pH could index NAE, regression analyses with 95 percent prediction intervals were used. Independent samples was assumed and prior checking occurred for linearity, independence of error terms (Durbin-Watson), normality of residuals, heteroscedasticity and outliers (+/->3 SD). To assess the ability of spot-tests (last evening and first morning) to track changes following NEAP modulation, a two way ANOVA with repeated measures (days X treatment) was used. Data was checked for normality, outliers (+/->3 studentised residuals) and sphericity (Mauchly’s test). In the instance a participants food intake drifted resulting in a PRAL deviation of >3 mEq on any day during cross over, the participant was excluded from the ANOVA and consequently their case was removed. Non-normal data was transformed by square root and again, assumptions checked. Effects sizes were calculated as partial eta squared (?2). Additionally, the average of the maximum and minimum spot-tests on the days where all voids were spot-tested is reported, as is the percent of participants whom had maximum and minimum pH units >1.5 pH units apart.

To assess the capacity of the supplements to modulate NEAP, paired t-tests were computed for the primary (24-hr NAE and urine pH) endpoints. In addition such analysis was carried out on the second morning spot-test. For these analyses, only participants whose intake drifted (>3 mEq) on the day of the 24-hr collections were excluded. Outliers (by boxplot) and normality of difference scores was checked, while, effects sizes were calculated as Cohen’s d. These statistical analyses were completed using Microsoft Excel (XP professional edition; Microsoft Corp, Redmond, USA), SPSS version 11 (SPSS Inc, Chicago, USA) and MedCalc (MedCalc Software, MedCalc, Mariakerke, Belgium). Summary data is presented as mean +/- SD. Two-tailed significance was accepted at P equal to or less than 0.05 and the Shapiro Wilk test was used to accept normality at P equal to or greater than 0.05.

To assess the agreement between estimated and analysed NEAP, the Bland and Altman method with repeat (duplicate) non constant observations (i.e., one observation during the placebo period and one observation during the supplement period on the same participant) was used. Equations were computed using dietary data from the concurrent 24-hr collection periods. The distribution of the differences was checked and outliers (+/->3.0 standard deviations (SD) of the difference) excluded. As non-constant observations require equal replications, those with unequal replicates were excluded. A priori limits of agreement were set at +/-15 mEq/d as this would permit a reasonable estimation of NEAP in individuals and also account for imprecision in the criterion method. These statistical analyses were completed using MedCalc (MedCalc Software, MedCalc, Mariakerke, Belgium).

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
Actual
1/05/2015
Date of last participant enrolment
Anticipated
Actual
1/08/2015
Date of last data collection
Anticipated
Actual
Sample size
Target
16
Accrual to date
Final
16
Recruitment in Australia
Recruitment state(s)
QLD

Funding & Sponsors
Funding source category [1] 293216 0
University
Name [1] 293216 0
University of the Sunshine Coast
Country [1] 293216 0
Australia
Funding source category [2] 293226 0
Commercial sector/Industry
Name [2] 293226 0
Morlife
Country [2] 293226 0
Australia
Primary sponsor type
Individual
Name
Dr Gary Slater
Address
University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs QLD 4556
Country
Australia
Secondary sponsor category [1] 292016 0
None
Name [1] 292016 0
Address [1] 292016 0
Country [1] 292016 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 294697 0
University of the Sunshine Coast Human Ethics committee
Ethics committee address [1] 294697 0
Ethics committee country [1] 294697 0
Australia
Date submitted for ethics approval [1] 294697 0
14/11/2014
Approval date [1] 294697 0
27/03/2015
Ethics approval number [1] 294697 0
S/14/70

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes
Attachments [1] 821 821 0 0
/AnzctrAttachments/370412-Ethics approval notice form.pdf

Contacts
Principal investigator
Name 64706 0
Dr Gary Slater
Address 64706 0
University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs QLD 4556
Country 64706 0
Australia
Phone 64706 0
+61 7 5456 5078
Fax 64706 0
Email 64706 0
[email protected]
Contact person for public queries
Name 64707 0
Ben Parmenter
Address 64707 0
University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs QLD 4556
Country 64707 0
Australia
Phone 64707 0
+61 423 536 798
Fax 64707 0
Email 64707 0
[email protected]
Contact person for scientific queries
Name 64708 0
Ben Parmenter
Address 64708 0
University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs QLD 4556
Country 64708 0
Australia
Phone 64708 0
+61 42353 6798
Fax 64708 0
Email 64708 0
[email protected]

No information has been provided regarding IPD availability


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
SourceTitleYear of PublicationDOI
Dimensions AIImmunopotentiating Activity of Fucoidans and Relevance to Cancer Immunotherapy2023https://doi.org/10.3390/md21020128
N.B. These documents automatically identified may not have been verified by the study sponsor.